CN109966780B - Method for separating functional nanoparticles from aquatic shellfish cooking soup - Google Patents
Method for separating functional nanoparticles from aquatic shellfish cooking soup Download PDFInfo
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- B01D15/08—Selective adsorption, e.g. chromatography
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Abstract
The invention discloses a method for separating functional nanoparticles from aquatic shellfish cooking soup. The separation method comprises the following steps: thermally processing aquatic shellfish to prepare aquatic shellfish cooking soup; centrifuging the aquatic shellfish cooking soup to obtain supernatant; separating the supernatant by combining gel exclusion chromatography with a dynamic light scattering instrument to obtain functional nanoparticles in the aquatic shellfish cooking soup. The nano-particles can effectively load active ingredients such as taurine, ornithine, phytosterol and the like, and can be applied to medicines or foods for regulating body immunity and relevant liver diseases. The method has the advantages of quick separation, high yield, no damage to the sample, high resolution, safe and green process, wide application field and the like.
Description
Technical Field
The invention belongs to the technical field of food and nano materials, and particularly relates to a method for separating functional nano particles in aquatic shellfish cooking soup.
Background
Aquatic shellfish, such as Corbicula fluminea (Corbicula fluminea) is bivalve mollusk, the shell of which is yellow green, brown yellow, or black brown, belonging to Corbicula genus (Corbicula) of Heterodentata (Heterodonta) of Heterodentata of Eulamellibranchiales (Eulamellibranchia) of Lamellibranchia (Lamellibranchia), also called Corbicula fluminea, Scapharca granosa, Spirospermum, etc. Corbicula fluminea is one of important economic shellfish in China, and is widely distributed in warm and humid fresh water or brackish water river and lake environments, such as Zhejiang, Fujian, Jiangsu, Taiwan and other provinces in the southeast. The Corbicula fluminea soft part (Corbicula fluminea meat) is delicious in taste and has quite remarkable health care value.
Due to the great advantages brought by the nanometer size, the nanometer material is widely applied to various research fields, such as liposome, hydrogel, microemulsion and the like. The artificial nano materials have a long period from practical application due to the existence of organism safety inspection, and meanwhile, the practical benefits of the artificial nano materials need to be further verified due to the complex environment in the body. The functionality and safety of the micro-nano particles in traditional food such as corbicula fluminea soup are inspected by people for thousands of years, and the micro-nano particles have a wide market application prospect. The micro-nano particles in the corbicula fluminea soup have active ingredients such as taurine, ornithine, phytosterol and the like, namely the corbicula fluminea nano particles have the loading capacity of the active ingredients, can directly act with digestive tract macrophages, and can effectively reduce blood fat and protect liver. Therefore, how to separate these functional nanoparticles becomes a hot spot for researchers to study.
The applicant researches in advance that the nano particles in corbicula fluminea can be well separated by means of static multi-angle laser light scattering, size exclusion chromatography separation or ion exchange chromatography and the like, so that the functional nano particle freeze-dried powder is obtained. However, the above separation means still has disadvantages. For example, the sensitivity of static multi-angle laser light scattering is low, the manufacturing cost is expensive, and the separation cost is high; the time consumption of size exclusion chromatography is long, and the separation efficiency is low; the high-salt elution process of ion exchange chromatography may cause certain interference to systems other than corbicula fluminea, and the higher ionic strength is easy to cause the denaturation of some proteins and the loss of active ingredients and may not be suitable for the preservation of general samples.
Therefore, based on the defects of the prior separation method, the separation method for functional nanoparticles in the aquatic shellfish cooking soup is developed, and the separation method adopts gel exclusion chromatography combined with dynamic light scattering, so that the separation sensitivity is high, and the separation method has the advantages of low cost, high efficiency and the like.
Disclosure of Invention
The invention aims to provide a method for separating functional nanoparticles in aquatic shellfish cooking soup. In order to achieve the above purpose, the invention provides the following technical scheme:
a method for separating functional nanoparticles from aquatic shellfish cooking soup comprises the following steps:
(1) thermally processing aquatic shellfish to prepare aquatic shellfish cooking soup;
(2) centrifuging the aquatic shellfish cooking soup obtained in the step (1) to obtain supernatant;
(3) and (3) separating the supernatant obtained in the step (2) by combining gel exclusion chromatography with a dynamic light scattering instrument to obtain functional nano particles in the aquatic shellfish cooking soup.
Further, the aquatic shellfish includes corbicula fluminea, clam, mussel, and bivalve shellfish.
Further, the preparation process of the aquatic shellfish thermal processing comprises the following steps: cleaning aquatic shellfish with distilled water, standing for more than 5h, weighing to obtain fresh individual, adding distilled water according to the material-liquid ratio of 1:1-3kg/L, boiling for 0.5-2h, cooling to room temperature, and filtering with gauze to obtain aquatic shellfish cooking soup.
Further, the centrifugation speed in the step (2) is 5000--1And the centrifugation time is 10-20 min.
Further, the specific method in the step (3) is as follows: and introducing the supernatant into a gel exclusion chromatographic column to enable the functional nanoparticles to be fully combined on the gel exclusion chromatographic column, then eluting with a buffer solution, simultaneously monitoring the eluent by 280nm ultraviolet band combined online dynamic light scattering, and collecting the eluent with the common superposition peak of ultraviolet absorption and light scattering, thus obtaining the functional nanoparticles in the aquatic shellfish cooking soup.
Furthermore, the gel exclusion chromatographic column is agarose or a derivative cross-linked product thereof, the separation range is 60KDa-20000KDa, the pore diameter is 45-165 mu m, the tolerance flow rate is higher than that of a common gel column, and the main model is Sepharose CL-2B/4B/6B.
Further, the buffer eluent is phosphate buffer, the concentration of the phosphate buffer is 0.05M, and the pH value is 7.2.
Further, the particle size of the functional nanoparticles in the aquatic shellfish cooking soup is 40-150 nm.
Further, the recovery rate of the functional nanoparticles in the aquatic shellfish cooking soup by the separation method is higher than 90%.
The invention has the advantages that:
(1) the combined separation method provided by the invention is rapid, high in yield, mild in separation mode, free of damage to the sample, convenient to operate, green, high in safety and simple to prepare.
(2) The combined separation technology has good separation effect, and can obtain functional nano particles from complex food matrixes and effectively separate the functional nano particles from free components. Meanwhile, continuous separation can be carried out according to the size of the nano particles from large to small, the resolution can reach 1nm or even below, and the overall recovery rate of the nano particles is determined according to the total light scattering amount and can exceed 90 percent.
(3) The functional nanoparticles disclosed by the invention have active ingredients, also have the function of loading the active ingredients, can effectively load active products such as taurine and ornithine, and have loading rates of over 60%.
(4) The functional nano-particles obtained by separation can be applied to medicines or foods related to the regulation of body immunity and liver diseases, and have wide application prospects.
Drawings
Fig. 1 shows gel chromatography and on-line dynamic light scattering of functional nanoparticles (a is gel chromatography separation of supernatant of corbicula fluminea soup, solid line is chromatogram of ultraviolet wavelength of 280nm, dotted line is light scattering intensity, F1, F2 are particle components, and F3 is mainly free component).
FIG. 2 is TEM electron micrograph (A) and physical image (B) of functional nanoparticles.
Figure 3 is a linear plot of functional nanoparticle dilution concentration versus light scattering intensity.
Figure 4 functional nanoparticle component content.
Figure 5 loading of functional nanoparticle active ingredients.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
Example 1:
(1) cleaning corbicula fluminea with distilled water, standing for 5h, weighing fresh living individuals according to a feed-liquid ratio of 1 kg: adding 1L of the mixture into a stainless steel pot containing distilled water, placing the stainless steel pot on an induction cooker, boiling for 1h, cooling, and filtering with gauze;
(2) placing the coarse filtrate in a centrifuge at a centrifugation speed of 15000r min-1, and centrifuging at 4 deg.C for 15min to obtain supernatant;
(3) the supernatant was rapidly separated using a gel exclusion chromatography column (10 mm. times.120 mm) packed with Sepharose CL-4B, and the column was packed with 0.05M phosphate buffer (pH7.2) and equilibrated at the flow rate: 0.5mL/min, the loading amount is 1mL, and the peak appearance of the functional nanoparticles is detected by an ultraviolet band of 280nm combined online dynamic laser light scattering instrument (SEC-DLS) (see figure 1), which is different from the free protein component. The molecular species is collected in a plurality of tubes, each tube is 2mL, the common superposition peak of ultraviolet absorption and light scattering appears at 12-32min, the molecular species is functional nanoparticles, the molecular species can be further divided into two main nanoparticle subgroups, the average particle diameters of the two subgroups are 70.86 +/-1.82 nm and 45.88 +/-4.16 nm (namely F1 segment and F2 segment in figure 1), and the colloidal properties are shown in Table 1. The whole separation process is rapid, and the separation cost is low.
Example 2: SEC-DLS can continuously separate nano particles in corbicula fluminea soup and detect the nano size on line, so that a nano particle group with the size of 150-40nm can be obtained, and the resolution can reach 1 nm. The separation spectrum is shown in figure 1, the particle morphology is shown in figure 2, and the comparative reference Soup in figure 2 refers to the supernatant.
Example 3: compared with the original soup particles, the nano-particles obtained by SEC have no sharp increase or decrease of the nano-size, which indicates that the SEC separation conditions are milder. See fig. 3 and table 1, respectively.
Example 4: by utilizing the characteristic that the total light scattering amount of the nanoparticles in the corbicula fluminea soup changes linearly under different dilution times (4 times, 16 times, 64 times and 256 times), the recovery rate of the nanoparticles is calculated by measuring the light scattering, and the method in the whole process is quick and accurate.
Example 5: the corbicula fluminea soup contains high-content polysaccharide, the polysaccharide component in the corbicula fluminea soup is proved to have obvious antioxidation and immunoregulation functions, meanwhile, the nano particles in the corbicula fluminea soup are loaded with small-molecule active ingredients such as taurine, ornithine, phytosterol and the like, and the content is respectively 4.48 mu g/mg, 31.22 mu g/mg and 111 mu g/g through detection. The loading rate (the active ingredients in the corbicula fluminea particles account for the total active ingredient content in the soup) is over 60 percent. Referring specifically to fig. 4 and 5, the claum Soup in fig. 4 and 5 refers to the supernatant. These components have an immunomodulating effect on the one hand and are of relevance for the treatment of liver diseases on the other hand.
TABLE 1
Claims (8)
1. A method for separating functional nanoparticles from aquatic shellfish cooking soup is characterized by comprising the following steps:
(1) thermally processing aquatic shellfish to prepare aquatic shellfish cooking soup;
(2) centrifuging the aquatic shellfish cooking soup obtained in the step (1) to obtain supernatant;
(3) separating the supernatant obtained in the step (2) by combining gel exclusion chromatography with a dynamic light scattering instrument to obtain functional nanoparticles in the aquatic shellfish cooking soup;
the specific method of the step (3) is as follows: and introducing the supernatant into a gel exclusion chromatographic column to enable the functional nanoparticles to be fully combined on the gel exclusion chromatographic column, then eluting with a buffer solution, simultaneously monitoring the eluent by 280nm ultraviolet band combined online dynamic light scattering, and collecting the eluent with the common superposition peak of ultraviolet absorption and light scattering, thus obtaining the functional nanoparticles in the aquatic shellfish cooking soup.
2. The separation method of claim 1, wherein the aquatic shellfish comprises corbicula fluminea, clam, mussel, arctic shell bivalve.
3. The separation method according to claim 1, wherein the aquatic shellfish thermal processing is prepared by: cleaning aquatic shellfish with distilled water, standing for more than 5h, weighing to obtain fresh individual, adding distilled water according to the material-liquid ratio of 1:1-3kg/L, boiling for 0.5-2h, cooling to room temperature, and filtering with gauze to obtain aquatic shellfish cooking soup.
4. The separation method according to claim 1, wherein the centrifugation speed in step (2) is 5000-10000 r-min-1And the centrifugation time is 10-20 min.
5. The separation method according to claim 1, wherein the gel exclusion chromatography column packing is agarose or a derivative cross-linked product thereof, the separation range is 60KDa to 20000KDa, and the pore size is 45 to 165 μm.
6. The separation process according to claim 1, wherein the buffered eluent is a phosphate buffer at a concentration of 0.05M and a pH of 7.2.
7. The separation method as claimed in claim 1, wherein the particle size of the functional nanoparticles in the aquatic shellfish cooking soup is 40-150 nm.
8. The separation method of claim 1, wherein the recovery rate of the functional nanoparticles in the aquatic shellfish cooking soup is higher than 90%.
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